The underlying mechanisms responsible for the control of cardiac contractility will continue to be investigated. Emphasis will be placed upon understanding the relationships which exist in myocardial tissue between cyclic AMP, cyclic GMP and calcium ion during normal contraction and during the positive and negative inotropic actions of isoproterenol and acetylcholine, respectively. The recent observation, made during the course of the project, that the positive inotropic effect of isoproterenol is sodium dependent but that the ability of isoproterenol to decrease the time to peak tension development is unaltered in contracting guinea pig atria in sodium free solutions, will be further investigated. The ability of isoproterenol to elevate cyclic AMP is reduced in sodium free solutions and the mechanisms involved will be investigated. This should further our understanding about the sites of isoproterenol action and the role of cyclic AMP in its inotropic action. Cyclic AMP, GMP and calcium metabolism will be measured in tissues treated with isoproterenol and acetylcholine. Free-bound tissue cyclic AMP will be quantitated by methods developed in this laboratory in an effort to identify the site of the defect in cyclic AMP production. Transmembrane action potentials will be measured using glass microelectrodes to determine whether the absence of sodium eliminates the inotropic action of isoproterenol by preventing the slow calcium currents which occur with isoproterenol in normal sodium containing solutions. These experiments will be extended to studies wherein these relationships are explored during single contraction cycles of the myocardium. A second aspect of this project is to determine the mechanism of catecholamine refractoriness which occurs in a rat astrocytoma cell line. Our previous studies indicate that rapid protein synthesis is involved in the refractoriness since cycloheximide and actinomycin D can prevent or reverse refractoriness. Studies will be aimed at further characterization and elucidation of refractoriness at the cellular and adenylate cyclase sites.